鸡p15～（ink4b）基因克隆、表达及生物学作用的研究
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摘要
INK4a_ARF基因座在控制细胞增殖和肿瘤抑制方面有着非常重要的作用,它在控制各种家族性和单发生性肿瘤方面发挥着十分重要的作用。INK4b和INK4a在人类染色体9p21上串联排列,在小鼠和大鼠中分别位于4号和5号染色体上。P16因子是CDK4/6-cyclinD的抑制物,主要是抑制CDK4/6-cyclinD复合物(complex)在细胞周期G1期晚期磷酸化PRb蛋白,使细胞不能通过G1/S检测点。大量研究表明INK4a/4b是由一个基因重复而来。在河豚中发现只存在INK4b,因此INK4b相对来说比INK4a更原始。INK4a基因能够编码两个结构和功能都不相同的产物。两个转录产物分别命名为α和β,它们是利用不同的启动子从不同的起始位点开始转录并利用不同的外显1α和1β和同样的外显子2,3剪接而成。α转录物被翻译为P16,β转录物被翻译为P14~(ARF)(小鼠中为P19~(ARF))。P16在PRb上游起作用, ARF在P53上游起作用,它与MDM2直接结合,防止MDM2介导P53降解。2002年Soo-Hyun等人从鸡基因组的部分酶切片段中克隆到完整的p15~(ink4b)基因和截短的ARF基因,但没有发现p16~(ink4a)基因,由此可猜想在没有P16因子的鸡细胞内,P15因子在鸡细胞周期的调控中可能发挥着更重要的作用。
本研究首先构建了pVAX1-p15~(ink4b)基因的真核表达载体,将其转染进鸡胚成纤维细胞(CEFs)。RT-PCR和westernblot检测p15~(ink4b)在CEFs内转录和表达情况。然后就p15~(ink4b)对CEFs的增殖和衰老相关性β-半乳糖苷酶影响进行了研究。结果表明,p15~(ink4b)在CEFs中能够转录和表达,抑制CEFs的增殖,将CEFs抑制在细胞周期G1/S期检测点上,能够诱导CEFs进入细胞衰老。
总之,本研究首次对鸡p15~(ink4b)对细胞周期调控和诱导细胞衰老的生物学功能进行了初步的探讨,为进一步研究p15~(ink4b)生物学作用,细胞周期的调控机理和肿瘤的发生机理奠定了基础。
INK4a_ARF locus has a crucial role on controlling cell proliferation and tumor suppression.It can control a variety of familial and monogenesis tumors.INK4b and INK4a locate on human chromosome 9p21 in tandem(Human Genome Organization designation CDKN2B-CDKN2A).In mice and rat counterpart , they locate on chromosome 4 and chromosome 5 respectively.As an inhibitor of complex CDK4/6-cyclinD, P16 inhibits phosphrating PRb by CDK4/6-cyclinD in late G1.Thus,cell can not bypass G1/S restriction point.Accumulating research evidence indicates that INK4a/4b evolves from gene repetition.Gene INK4a evolves from gene INK4b.Thus, gene INK4b is more primitive than gene INK4a .Soo-Hyun and his parterners have isolated and characterized the equivalent locus in chickens in 2002.Surprisingly, they found that orthologues of INK4b and ARF, chickens do not encode an equivalent of INK4a.From above discussion,we can make hypothesis that P15 have a strong role in non-P16 chicken cells.As follows,this research was divided two parts.
Cloning and expression chicken p15~(ink4b) gene
The chicken p15~(ink4b) gene was amplified by PCR, then the PCR product and vector pVAX1 digest with double enzyme BamHI and XhoI, chicken p15~(ink4b) gene was cloned into a eukaryotic expression vector pVAX1.Then, we identifided positive clone by PCR and bienzymatic digestion and furtherly identified by sequencing.Sequencing result was analyzed by biological software. It was confirmed that the gene sequence was consistent with the sequence in GENEBANK.Positive recombinant eukaryotic plasmids were transfected into CEFs. We detected transcription and expression of p15~(ink4b) by employed RT-PCR and westernblot in CEFs.
Chicken p15~(ink4b) gene inhibited proliferation of CEFs, arrested CEFs in G1/G0 and induced cellular senescence of CEFs
We transfected pVAX1-p15 into CEFs by Lipofectamine2000. After transient transfection,proliferation of CEFs was determinated by direct cell number counting and MTT assay at 24, 48, 72 hours respectively. Cell cycle arrest was detected by fluorescence activated cell sorter (FACS) analysis.Cellular senescence was determinated by detecting SA-β- galactosidase. Results show that P15 can express in CEFs, nhibit cell cycle progression of CEFs at G1/S restriction point and induce cellular senescence CEFs.
In conclusion,this research explored biological activities of P15 in CEFs initially, which would lay a foundation to the researches in mechanism of cell senesence,cell cycle regulation and tumor genesis.
本研究首先构建了pVAX1-p15~(ink4b)基因的真核表达载体,将其转染进鸡胚成纤维细胞(CEFs)。RT-PCR和westernblot检测p15~(ink4b)在CEFs内转录和表达情况。然后就p15~(ink4b)对CEFs的增殖和衰老相关性β-半乳糖苷酶影响进行了研究。结果表明,p15~(ink4b)在CEFs中能够转录和表达,抑制CEFs的增殖,将CEFs抑制在细胞周期G1/S期检测点上,能够诱导CEFs进入细胞衰老。
总之,本研究首次对鸡p15~(ink4b)对细胞周期调控和诱导细胞衰老的生物学功能进行了初步的探讨,为进一步研究p15~(ink4b)生物学作用,细胞周期的调控机理和肿瘤的发生机理奠定了基础。
INK4a_ARF locus has a crucial role on controlling cell proliferation and tumor suppression.It can control a variety of familial and monogenesis tumors.INK4b and INK4a locate on human chromosome 9p21 in tandem(Human Genome Organization designation CDKN2B-CDKN2A).In mice and rat counterpart , they locate on chromosome 4 and chromosome 5 respectively.As an inhibitor of complex CDK4/6-cyclinD, P16 inhibits phosphrating PRb by CDK4/6-cyclinD in late G1.Thus,cell can not bypass G1/S restriction point.Accumulating research evidence indicates that INK4a/4b evolves from gene repetition.Gene INK4a evolves from gene INK4b.Thus, gene INK4b is more primitive than gene INK4a .Soo-Hyun and his parterners have isolated and characterized the equivalent locus in chickens in 2002.Surprisingly, they found that orthologues of INK4b and ARF, chickens do not encode an equivalent of INK4a.From above discussion,we can make hypothesis that P15 have a strong role in non-P16 chicken cells.As follows,this research was divided two parts.
Cloning and expression chicken p15~(ink4b) gene
The chicken p15~(ink4b) gene was amplified by PCR, then the PCR product and vector pVAX1 digest with double enzyme BamHI and XhoI, chicken p15~(ink4b) gene was cloned into a eukaryotic expression vector pVAX1.Then, we identifided positive clone by PCR and bienzymatic digestion and furtherly identified by sequencing.Sequencing result was analyzed by biological software. It was confirmed that the gene sequence was consistent with the sequence in GENEBANK.Positive recombinant eukaryotic plasmids were transfected into CEFs. We detected transcription and expression of p15~(ink4b) by employed RT-PCR and westernblot in CEFs.
Chicken p15~(ink4b) gene inhibited proliferation of CEFs, arrested CEFs in G1/G0 and induced cellular senescence of CEFs
We transfected pVAX1-p15 into CEFs by Lipofectamine2000. After transient transfection,proliferation of CEFs was determinated by direct cell number counting and MTT assay at 24, 48, 72 hours respectively. Cell cycle arrest was detected by fluorescence activated cell sorter (FACS) analysis.Cellular senescence was determinated by detecting SA-β- galactosidase. Results show that P15 can express in CEFs, nhibit cell cycle progression of CEFs at G1/S restriction point and induce cellular senescence CEFs.
In conclusion,this research explored biological activities of P15 in CEFs initially, which would lay a foundation to the researches in mechanism of cell senesence,cell cycle regulation and tumor genesis.
引文
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